Studies on mineral formation within extracellular matrix vesicles in healing rachitic rats epiphyseal cartilages have been completed, in respect to electron microscopic observations of cartilage. During this last year it has been possible, with micro knives, to cut out isolated calcifying septa free of cells and to study these in incubating synthetic lymphs. A new method of zonal centrifugation as well as a method of separating glycoproteins in guanidinium-casium chloride gradients on an ultramicro scale have been developed. A profile of polydispersity of proteoglycans in the puncture fluids from calcifying sites has been made with a new transport method of analytical ultracentrifugation. The heavier half of the aggregates fraction has been shown to be an inhibitor of mineral growth and susceptible to degradation to subunits by cartilage lysozyme (in studies collaborating with Dr. Klaus Kuettner). The nucleational agent has been localized to 1/6 fraction of a capillary tube column by zonal centrifugation. This nucleating material has a high alkaline phosphatase and at present is considered to contain vesicle membranes. Future studies will be to further delineate the nature of the nucleational agent by EM and by biochemical techniques.